1. The Field of the Invention
The present invention relates to assay methods to predict compound toxicity.
2. The Relevant Technology
The comet assay, also known as Single Cell Gel Electrophoresis (SCGE), is a standard assay commonly used to measure the induction of DNA damage by acute exposure to chemicals or radiation. The comet assay was first described by Singh et al. in 1988. It has since gained in popularity as a standard technique for evaluation of DNA damage/repair, biomonitoring and genotoxicity testing. A comet assay involves the encapsulation of cells in a low-melting-point agarose (LMA) suspension, lysis of the cells in neutral or alkaline conditions to break down the cell membranes and leave the nuclear area (or nucleoid), and electrophoresis of the layered nucleoids. This is followed by visual analysis with staining of DNA and calculating fluorescence to determine the extent of DNA damage.
The concept underlying the comet assay is that undamaged DNA retains a highly organized association with matrix proteins in the nucleus. When damaged, this organization is disrupted. The individual strands of DNA lose their compact structure and relax, expanding out of the cavity into the agarose. When an electric field is applied across the DNA, which has an overall negative charge, the DNA is drawn towards the anode, which is positively charged. Undamaged DNA strands are too large and do not leave the cavity, whereas damaged fragments do. The smaller the fragments, the farther they are free to move in a given period of time. Therefore, the amount of DNA that leaves the cavity is a measure of the amount of DNA damage in the cell.
The image analysis measures the overall intensity of the fluorescence for the whole nucleoid and the fluorescence of the migrated DNA and compares the two signals. The stronger the signal from the migrated DNA, the more damage there is present. The overall structure resembles a comet (hence “comet” assay) with a circular head corresponding to the undamaged DNA that remains in the cavity and a tail of damaged DNA. The brighter and longer the tail, the higher the level of damage.
The comet assay is a versatile technique for detecting damage and with adjustments to the protocol can be used to quantify the presence of a wide variety of DNA altering lesions (damage). The damage usually detected is single strand breaks and double strand breaks. Additional DNA structures can be detected as DNA damage, such as AP sites (abasic sites missing either a pyrimidine or purine nucleotide) and sites where excision repair is taking place.
Conventionally, the cells are encapsulated on a slide that contains one or more wells. Lysis occurs by placing the slide into a container and exposing the slide to a lysing reagent. Containers used in conventional comet assays may require up to a liter of lysing reagent to cover the slide. The slide is then transferred to an electrophoresis chamber, where the slide is exposed to an electrophoresis buffer solution and an electric current is passed over the slide. Conventional electrophoresis chambers may require up to a liter of electrophoresis buffer solution to cover the slide. The slide is then removed from the electrophoresis chamber, stained with a fluorescent dye, and imaged through a microscope to determine the extent of DNA damage. This is performed by manual scoring by eye or automatically by imaging software.
While conventional assaying systems provide acceptable results when used in performing comet assays, the systems have some significant problems. For example, as noted above, up to a liter of lysing reagent and a liter of electrophoresis buffer are often required to carry out a comet assay on a single slide. After use, these solutions must be discarded. However, the solutions are not environmentally friendly, and special care must be taken in their disposal. It would be a great benefit to provide an assaying system that minimized the use of these solutions. Another problem with the present approach is that slides must be moved between different containers to perform the steps of the comet assay. The cells that are encapsulated on the slide can be very delicate and affected by the movement. Therefore, it would be a benefit to minimize the movement of the slide between different containers. Furthermore, by requiring movement between different containers, the conventional assaying system does not lend itself well to automated or robotic handling of the slides during the comet assay.